Guillain-Barré syndrome (GBS) is an autoimmune disorder that causes weakness, sensory loss, autonomic dysfunction, and chronic neuropathic pain. The mediators responsible for driving early autoimmune injury in the most common GBS variant, acute inflammatory demyelinating polyradiculoneuropathy (AIDP), remain incompletely understood. We performed bulk and single-cell RNA sequencing on peripheral blood mononuclear cells collected from early untreated AIDP-variant GBS patients and healthy controls to comprehensively deduce leukocyte transcriptome alterations and predict disease-driving interactions between pathogenic leukocytes and peripheral nervous system cells. Bulk transcriptomics showed interferon- and JAK/STAT-driven proinflammatory signatures in myeloid cells. CD8+ T-cells were highly proliferative and expressed axon guidance genes. Single-cell transcriptomics revealed dysregulation of CD4+FOXP3+ regulatory and CD4+ effector memory T-cells indicative of poorly controlled type 1 and type 17 immune responses. Highly activated intermediate monocytes upregulated genes related to angiogenesis and oncostatin M. Interactome analysis between GBS leukocytes, Schwann cells, and sensory neurons predicted increased engagement of ligand-receptor pairs with nerve integrity and pain functions, including epiregulin, interferon-beta, adrenomedullin, clusterin, IL-6, IL-15, and CCL4. Functional validation demonstrated that CCL4 sensitizes human sensory neurons in vitro. These results unearth molecular interactions by which specific leukocyte populations in AIDP-variant GBS may induce peripheral nerve injury and drive neuropathic pain.

Diabetic peripheral neuropathy (DPN) is a prevalent complication of diabetes mellitus caused by metabolic toxicity to peripheral axons. We aimed to gain deep mechanistic insight into the disease using transcriptomics on tibial and sural nerves recovered from lower leg amputations in a mostly diabetic population and control sural nerves from cross-facial nerve graft surgery. First, comparing DPN versus control sural nerves revealed inflammatory activation and sensory changes in DPN. Second, when comparing mixed sensory and motor tibial and purely sensory sural nerves, we identified key pathway differences in affected DPN nerves, with distinct immunological features observed in sural nerves. Third, spatial transcriptomics of sural nerves revealed shifts in immune cell types associated with axonal loss progression. We also found clear evidence of neuronal transcript changes, like PRPH, in nerves with axonal loss, suggesting perturbed RNA transport into distal sensory axons. This motivated further investigation into neuronal mRNA localization in peripheral nerve axons, generating evidence of robust localization of mRNAs such as SCN9A and TRPV1 in human sensory axons. Our work provides insight into altered cellular and transcriptomic profiles in human nerves in DPN and highlights sensory axon mRNA transport as a potential contributor to nerve degeneration.

Mouse models are invaluable to understanding fundamental mechanisms in vascular biology during development, in health and different disease states. Several constitutive or inducible models that selectively knockout or knock in genes in vascular endothelial cells exist; however, functional and phenotypic differences exist between microvascular and macrovascular endothelial cells in different organs. In order to study microvascular endothelial cell-specific biological processes, we developed a Tamoxifen-inducible von Willebrand Factor (vWF) Cre recombinase mouse in the SJL background. The transgene consists of the human vWF promoter with the microvascular endothelial cell-selective 734 base pair sequence to drive Cre recombinase fused to a mutant estrogen ligand-binding domain [ERT2] that requires Tamoxifen for activity (CreERT2) followed by a polyadenylation (polyA) signal. We initially observed Tamoxifen-inducible restricted bone marrow megakaryocyte and sciatic nerve microvascular endothelial cell Cre recombinase expression in offspring of a mixed strain hemizygous C57BL/6-SJL founder mouse bred with mT/mG mice, with >90% bone marrow megakaryocyte expression efficiency. Founder mouse offspring were backcrossed to the SJL background by speed congenics, and intercrossed for >10 generations to develop hemizygous Tamoxifen-inducible vWF Cre recombinase (vWF-iCre/+) SJL mice with stable transgene insertion in chromosome 1. Microvascular endothelial cell-specific Cre recombinase expression occurred in the sciatic nerves, brains, spleens, kidneys and gastrocnemius muscles of adult vWF-iCre/+ SJL mice bred with Ai14 mice, with retained low level bone marrow and splenic megakaryocyte expression. This novel mouse strain would support hypothesis-driven mechanistic studies to decipher the role(s) of specific genes transcribed by microvascular endothelial cells during development, as well as in physiologic and pathophysiologic states in an organ- and time-dependent manner.

Diabetic peripheral neuropathy (DPN) is a prevalent complication of diabetes mellitus that is caused by metabolic toxicity to peripheral axons. We aimed to gain deep mechanistic insight into the disease process using bulk and spatial RNA sequencing on tibial and sural nerves recovered from lower leg amputations in a mostly diabetic population. First, our approach comparing mixed sensory and motor tibial and purely sensory sural nerves shows key pathway differences in affected nerves, with distinct immunological features observed in sural nerves. Second, spatial transcriptomics analysis of sural nerves reveals substantial shifts in endothelial and immune cell types associated with severe axonal loss. We also find clear evidence of neuronal gene transcript changes, like PRPH, in nerves with axonal loss suggesting perturbed RNA transport into distal sensory axons. This motivated further investigation into neuronal mRNA localization in peripheral nerve axons generating clear evidence of robust localization of mRNAs such as SCN9A and TRPV1 in human sensory axons. Our work gives new insight into the altered cellular and transcriptomic profiles in human nerves in DPN and highlights the importance of sensory axon mRNA transport as an unappreciated potential contributor to peripheral nerve degeneration.

INTRODUCTION/AIMS:The precise relationship between molecular mimicry and tissue-specific autoimmunity is unknown. Major histocompatibility complex (MHC) class II antigen presenting cell-CD4+ T-cell receptor complex interactions are necessary for adaptive immunity. This study aimed to determine the role of endoneurial endothelial cell MHC class II in autoimmune polyneuropathy. METHODS:Cryopreserved Guillain-Barré syndrome (GBS) patient sural nerve biopsies and sciatic nerves from the severe murine experimental autoimmune neuritis (sm-EAN) GBS model were studied. Cultured conditional ready MHC Class II antigen A-alpha chain (H2-Aa) embryonic stem cells were used to generate H2-Aaflox/+ C57BL/6 mice. Mice were backcrossed and intercrossed to the SJL background to generate H2-Aaflox/flox SJL mice, bred with hemizygous Tamoxifen-inducible von Willebrand factor Cre recombinase (vWF-iCre/+) SJL mice to generate H2-Aaflox/flox; vWF-iCre/+ mice to study microvascular endothelial cell adaptive immune responses. Sm-EAN was induced in Tamoxifen-treated H2-Aaflox/flox; vWF-iCre/+, H2-Aaflox/flox; +/+, H2-Aa+/+; vWF-iCre/+ and untreated H2-Aaflox/flox; vWF-iCre/+ adult female SJL mice. Neurobehavioral, electrophysiological and histopathological assessments were performed at predefined time points. RESULTS:Endoneurial endothelial cell MHC class II expression was observed in normal and inflamed human and mouse peripheral nerves. Tamoxifen-treated H2-Aaflox/flox; vWF-iCre/+ mice were resistant to sm-EAN despite extensive MHC class II expression in lymphoid and non-lymphoid tissues. DISCUSSION:A conditional MHC class II knockout mouse to study cell- and time-dependent adaptive immune responses in vivo was developed. Initial studies show microvascular endothelial cell MHC class II expression is necessary for peripheral nerve specific autoimmunity, as advocated by human in vitro adaptive immunity and ex vivo transplant rejection studies.

Immune-mediated demyelinating polyneuropathies (IMDPs) are rare disorders in which dysregulated adaptive immune responses cause peripheral nerve demyelinating inflammation and axonal injury in susceptible individuals. Despite significant advances in understanding IMDP pathogenesis guided by patient data and representative mammalian models, specific therapies are lacking. Significant knowledge gaps in IMDP pathogenesis still exist, e.g. precise antigen(s) and mechanisms that initially trigger immune system activation and identification of large population disease susceptibility factors. The initial directional cues for antigen-specific effector or autoreactive leukocyte trafficking into peripheral nerves are also unknown. An overview of current animal models, with emphasis on the experimental autoimmune neuritis and spontaneous autoimmune peripheral polyneuropathy models, is provided. Insights on the initial directional cues for peripheral nerve tissue specific autoimmunity using a novel Major Histocompatibility Complex class II conditional knockout mouse strain are also discussed, suggesting an essential research tool to study cell- and time-dependent adaptive immunity in autoimmune diseases.

Background Mitochondrial cardiomyopathy (MCM) is an alteration in cardiac structure and function caused by gene mutations or deletions affecting components of the mitochondrial respiratory chain. We report a case of MCM presenting as cardiogenic shock, ultimately requiring left ventricular assist device (LVAD) placement. Case summary A 35-year-old woman with chronic weakness and non-ischaemic cardiomyopathy, on home dobutamine, was referred to our institution for heart transplantation evaluation. She was admitted to the hospital for suspected cardiogenic shock after laboratory tests revealed a lactate level of 5.4 mmol/L (ref: 0.5-2.2 mmol/L). Her hospital course was complicated by persistently undulating lactate levels (0.2-8.6 mmol/L) that increased with exertion and did not correlate with mixed venous oxygen saturation measurements obtained from a pulmonary artery catheter. Electrodiagnostic testing demonstrated a proximal appendicular and axial myopathy. A left deltoid muscle biopsy was performed that demonstrated evidence of a mitochondrial disease on light and electron microscopy. Muscle genetic testing revealed two large-scale mitochondrial deoxyribonucleic acid sequence deletions, confirming the diagnosis of MCM. She subsequently underwent LVAD placement, which was complicated by significant right ventricular failure requiring early mechanical support. She was ultimately discharged home with chronic inotropic support. Discussion Mitochondrial cardiomyopathy in adults is a diagnostic and therapeutic challenge. Prompt diagnosis should be made in patients with unknown causes of heart failure via skeletal muscle histopathology guided by electrodiagnostic studies, and targeted genetic testing in affected tissue. Outcomes in adult MCM patients who receive an LVAD are unknown and warrant further investigation.

Drug delivery into the peripheral nerves and nerve roots has important implications for effective local anesthesia and treatment of peripheral neuropathies and chronic neuropathic pain. Similar to drugs that need to cross the blood–brain barrier (BBB) and blood–spinal cord barrier to gain access to the central nervous system (CNS), drugs must cross the peripheral nerve barriers (PNBs), formed by the perineurium and blood–nerve barrier to modulate peripheral axons. Despite significant progress made to develop effective strategies to enhance BBB permeability in therapeutic drug design, efforts to enhance drug permeability and retention in peripheral nerves and nerve roots are relatively understudied. Guided by knowledge describing structural, molecular, and functional similarities between restrictive neural barriers in the CNS and peripheral nervous system, it is hypothesized that certain CNS drug delivery strategies are adaptable for peripheral nerve drug delivery. Here, the molecular, structural, and functional similarities and differences between the BBB and PNB are described, existing CNS and peripheral nerve drug delivery strategies are summarized and compared, and the potential application of selected CNS delivery strategies to improve efficacious drug entry for peripheral nerve disorders is discussed.

Introduction:Major histocompatibility complex (MHC) class II professional antigen presenting cell-naïve CD4+ T cell interactions via the T-cell receptor complex are necessary for adaptive immunity. MHC class II upregulation in multiple cell types occurs in human autoimmune polyneuropathy patient biopsies, necessitating studies to ascertain cellular signaling pathways required for tissue-specific autoimmunity. Methods:Cryopreserved Guillain-Barré syndrome (GBS) patient sural nerve biopsies and sciatic nerves from the severe murine experimental autoimmune neuritis (sm-EAN) GBS model were studied. Cultured conditional ready MHC Class II antigen A-alpha chain (H2-Aa) embryonic stem cells were used to generate H2-Aa flox/+ C57BL/6 mice. Mice were backcrossed and intercrossed to the SJL background to generate H2-Aa flox/flox SJL mice, bred with hemizygous Tamoxifen-inducible von Willebrand factor Cre recombinase (vWF-iCre/+) SJL mice to generate H2-Aa flox/flox ; vWF-iCre/+ to study microvascular endothelial cell adaptive immune responses. Sm-EAN was induced in adult female SJL Tamoxifen-treated H2-Aa flox/flox ; vWF-iCre/+ mice and H2-Aa flox/flox ; +/+ littermate controls. Neurobehavioral, electrophysiological and histopathological assessments were performed at predefined time points. Results:Endoneurial endothelial cell MHC class II expression was observed in normal and inflamed human and mouse peripheral nerves. Adult female Tamoxifen-treated H2-Aa flox/flox ; vWF-iCre/+ did not develop sm-EAN despite extensive MHC class II expression in lymphoid and non-lymphoid tissues. Discussion:A conditional MHC class II knockout mouse to study cell- and time-dependent adaptive immune responses in vivo is developed. Initial studies show microvascular endothelial cell MHC class II expression is necessary for peripheral nerve specific autoimmunity, as advocated by human in vitro adaptive immunity and ex vivo transplant rejection studies.

Dynamin 2 mutations are associated with Charcot-Marie-Tooth neuropathy. We report two siblings with a novel missense heterozygous point mutation (c.1609 G>A) in the highly conserved pleckstrin homology domain in exon 15 of Dynamin 2 presenting with progressive length-dependent sensorimotor polyneuropathy with mixed demyelinating and axonal features on electrodiagnostic studies. The previously unrecognized missense point mutation, which was inherited from their symptomatic but previously undiagnosed mother, was determined to be likely pathogenic based on a non-conservative amino acid substitution (p.Gly537Ser) that is predicted to damage secondary protein structure or function. This report emphasizes the importance of recognizing inherited neuropathies in clinical practice and evaluating suspected pathogenic gene variants initially classified to be of undetermined clinical significance in family cohorts. These cases add to the spectrum of pathogenic Dynamin 2 mutations associated with dominant-intermediate Charcot-Marie-Tooth neuropathy.

Endosomal sorting plays a fundamental role in directing neural development. By altering the temporal and spatial distribution of membrane receptors, endosomes regulate signaling pathways that control the differentiation and function of neural cells. Several genes linked to inherited demyelinating peripheral neuropathies, known as Charcot-Marie-Tooth (CMT) disease, encode proteins that directly interact with components of the endosomal sorting complex required for transport (ESCRT). Our previous studies demonstrated that a point mutation in the ESCRT component hepatocyte growth-factor-regulated tyrosine kinase substrate (HGS), an endosomal scaffolding protein that identifies internalized cargo to be sorted by the endosome, causes a peripheral neuropathy in the neurodevelopmentally impaired teetering mice. Here, we constructed a Schwann cell-specific deletion of Hgs to determine the role of endosomal sorting during myelination. Inactivation of HGS in Schwann cells resulted in motor and sensory deficits, slowed nerve conduction velocities, delayed myelination and hypomyelinated axons, all of which occur in demyelinating forms of CMT. Consistent with a delay in Schwann cell maturation, HGS-deficient sciatic nerves displayed increased mRNA levels for several promyelinating genes and decreased mRNA levels for genes that serve as markers of myelinating Schwann cells. Loss of HGS also altered the abundance and activation of the ERBB2/3 receptors, which are essential for Schwann cell development. We therefore hypothesize that HGS plays a critical role in endosomal sorting of the ERBB2/3 receptors during Schwann cell maturation, which further implicates endosomal dysfunction in inherited peripheral neuropathies.SIGNIFICANCE STATEMENT Schwann cells myelinate peripheral axons, and defects in Schwann cell function cause inherited demyelinating peripheral neuropathies known as CMT. Although many CMT-linked mutations are in genes that encode putative endosomal proteins, little is known about the requirements of endosomal sorting during myelination. In this study, we demonstrate that loss of HGS disrupts the endosomal sorting pathway in Schwann cells, resulting in hypomyelination, aberrant myelin sheaths, and impairment of the ERBB2/3 receptor pathway. These findings suggest that defective endosomal trafficking of internalized cell surface receptors may be a common mechanism contributing to demyelinating CMT.

Leukocyte infiltration and persistence within peripheral nerves have been implicated in chronic nociception pathogenesis in murine peripheral neuropathy models. Endoneurial cytokine and chemokine expression contribute to leukocyte infiltration and maintenance of a pro-inflammatory state that delays peripheral nerve recovery and promotes chronic pain behaviors in these mice. However, there has been a failure to translate murine model data into safe and effective treatments for chronic neuropathic pain in peripheral neuropathy patients, or develop reliable biomarkers that may help diagnose or determine treatment responses in affected patients. Initial work showed that persistent sciatic nerve CD11b+ CD45+ leukocyte infiltration was associated with disease severity in three mouse models of inflammatory and traumatic peripheral neuropathies, implying a direct contributing role in disease pathogenesis. In support of this, CD11b+ leukocytes were also seen in the sural nerve biopsies of chronic neuropathic pain patients with three different peripheral neuropathies. Systemic CD11b antagonism using a validated function-neutralizing monoclonal antibody effectively treated chronic nociception following unilateral sciatic nerve crush injury (a representative traumatic neuropathy model associated with axonal degeneration and increased blood-nerve barrier permeability) and does not cause drug addiction behaviors in adult mice. These data suggest that CD11b could be an effective molecular target for chronic neuropathic pain treatment in inflammatory and traumatic peripheral neuropathies. Despite known murine peripheral neuropathy model limitations, our initial work suggests that early expression of pro-inflammatory cytokines, such as tissue inhibitor of metalloproteinases-1 may predict subsequent chronic nociception development following unilateral sciatic nerve crush injury. Studies aligning animal model investigation with observational data from well-characterized human peripheral neuropathies, including transcriptomics and proteomics, as well as animal model studies using a human clinical trial design should foster the identification of clinically relevant biomarkers and effective targeted treatments with limited addiction potential for chronic neuropathic pain in peripheral neuropathy patients.

Case Presentation: A 35-year-old woman with nonischemic cardiomyopathy, with a left ventricular ejection fraction <20% on home dobutamine, and chronic weakness was referred to our institution for heart transplantation evaluation. She was directly admitted to the hospital for suspected cardiogenic shock after her labs revealed a lactate level of 5.4 mmol/L. Her hospital course was complicated by persistently undulating lactate levels (0.2 - 8.6 mmol/L) that increased with exertion, despite normal mixed venous oxygen saturation measurements obtained from a pulmonary artery catheter. Serologic genetic testing was negative, and electromyography revealed the presence of a proximal and axial myopathy. A deltoid muscle biopsy was obtained, and genetic testing of the specimen revealed two mitochondrial deoxyribonucleic acid deletions, and electron microscopy revealed mitochondrial paracrystalline inclusions. She subsequently underwent left ventricular assist device placement and was started on carnitine, arginine, creatine, thiamine, and coenzyme Q10 supplementation. Despite left ventricular assist device placement, the patient has continued to decline due to progressive right heart failure and is currently being considered for cardiac transplantation. Discussion: Mitochondrial cardiomyopathy (MCM) is an alteration in cardiac structure and function caused by genetic defects of the mitochondrial respiratory chain, in the absence of other causes of heart failure. The true prevalence of MCM is unknown. Clinical manifestations in those with MCM present on a spectrum, ranging from asymptomatic cases to those with signs and symptoms typical of heart failure. Extra-cardiac features commonly include chronic weakness and fatigue. Diagnosis is made with prompt genetic testing and microscopic evaluation of a skeletal muscle biopsy specimen. Outcomes in patients who receive a left ventricular assist device are unknown and warrant further investigation.

OBJECTIVE To determine whether rituximab is safe and potentially beneficial, warranting further investigation in an efficacy trial for acetylcholine receptor antibody-positive generalized MG (AChR-Ab+ gMG). METHODS The B-Cell Targeted Treatment in MG (BeatMG) study was a randomized, double-blind, placebo-controlled, multicenter phase-2 trial that utilized a futility design. Individuals 21-90 years of age, with AChR-Ab+ gMG (MG Foundation of America Class II-IV) and receiving prednisone ≥15 mg/day were eligible. The primary outcome was a measure of steroid-sparing effect, defined as the proportion achieving ≥75% reduction in mean daily prednisone dose in the 4-weeks prior to week 52 and with clinical improvement or no significant worsening as compared to the 4-week period prior to randomization. The co-primary outcome was safety. Secondary outcomes included MG-specific clinical assessments. Fifty-two individuals were randomized (1:1) to either a two-cycle rituximab/placebo regimen, with follow-up through 52-weeks. RESULTS Of the 52 participants included, mean (±SD) age at enrollment was 55.1 (±17.1) years; 23 (44.2%) were female, and 31 (59.6%) were MGFA Class II. The mean (±SD) baseline prednisone dose was 22.1 (±9.7) mg/day. The primary steroid-sparing outcome was achieved in 60% of those on rituximab vs. 56% on placebo. The study reached its futility endpoint (p=0.03) suggesting that the pre-defined clinically meaningful improvement of 30% due to rituximab over placebo was unlikely to be achieved in a subsequent, larger trial. No safety issues identified. CONCLUSIONS While rituximab was safe and well-tolerated, these results suggest that there is a low probability of observing the defined clinically meaningful steroid-sparing effect over a 12-month period in a phase-3 trial of mild-moderately symptomatic AChR-Ab+ gMG. CLASSIFICATION OF EVIDENCE This study provides Class I evidence that for mild-to-moderate AChR-Ab+ gMG, compared with placebo, rituximab is safe but unlikely to reduce steroid use by an absolute difference of at least 30% at 1 year. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT02110706.

>Peripheral nerves coordinate signal transduction from the periphery to the central nervous system for processing and transmission back as required for normal mammalian function.Peripheral nerves and nerve roots are structurally divided into three compartments: the outermost epineurium,

Patients with progressive hand weakness may be seen in ambulatory medical clinics or in emergency rooms due to direct effects on activities of daily living or inadvertent injury associated with overuse or attempts to maintain normal function. It is important to recognize potential cause(s) and perform appropriate diagnostic tests and referrals that aid guide appropriate treatment that may lead to good outcomes. Hirayama disease is an underrecognized disorder in young adults due to an asymmetric growth-associated cervical spinal cord compression injury. Awareness of this disorder by internists, emergency room physicians, and radiologists would prevent unnecessary tests and interventions that may contribute to disease progression by delaying appropriate treatments or treating inappropriately, with consequential effects on outcomes. In this article, we describe 3 Hirayama disease cases from a single tertiary care institution and demonstrate how delayed diagnosis affected outcomes in 2 patients and early recognition facilitated improved outcomes in a patient.

Fibulin-5 (FBLN5) is an extracellular matrix glycoprotein expressed in elastic fiber-rich tissues.(1) Mutations affecting the first epidermal growth factor domain feature a spinal Charcot-Marie-Tooth (CMT) phenotype, whereas the C-terminus c.1117C>T variant causes demyelinating sensorimotor polyneuropathy.(2) Besides CMT, FBLN5 mutations also feature age-related macular degeneration and cutis laxa.(3) FBLN5-associated CMT was reported in 6 families from Austria,(2) the Czech Republic,(4) and China,(5) with 4 possessing the c.1117C>T (p.Arg373Cys) mutation.(2,4,5) Joint hyperlaxity and scoliosis were reported in 1 spinal CMT Austrian family due to a p.Gly90Ser mutation, but never with p.Arg373Cys-associated CMT-1.(2) We describe the phenotype of a family with FBLN5-associated CMT-1 featuring younger symptom onset, joint hyperlaxity, and scoliosis.